The long term goal of this continuing research program is to understand the activation of receptors and kinases on a molecular level in their native environment. A molecular understanding of receptor on-off switching, adaptation, and kinase regulation has not yet been achieved for any signaling pathway, despite the fact these events control essential cell processes in all organisms. Such understanding is crucial to a basic knowledge of biological signaling circuits, and to medical applications seeking to block or modify these circuits. The project focuses on the bacterial chemosensory pathway, which assembles to form an ultrasensitive, ultrastable, multi-protein array and is the prototype for the large superfamily of two-component signaling systems ubiquitous in bacteria and lower eukaroytes. In particular, the project examines an active, membrane-bound core complex that is the minimal functional unit of the full sensory array. This core complex contains the chemoreceptor, its associated histidine kinase CheA, and the coupling protein CheW. The Specific Aims will determine core complex architecture, will define the molecular mechanisms of receptor and kinase on-off switching, and will elucidate molecular interactions responsible for the ultrasensitivity and ultrastability of the signaling array. In addition, innovative site-directed cysteine chemistries and spectroscopic strategies are developed to probe structure and mechanism of the active signaling array in its native membrane environment.